Adenylate cyclase in many tissues is dually regulated, that is, subject to both stimulatory and inhibitory control. In eukaryotic cells, hormonal inhibition of adenylate cyclase is mediated by specific receptors and an inhibitory guanyl nucleotide-binding regulatory protein (Gi), whereas hormonal stimulation is mediated by receptors for stimulatory ligands and a stimulatory guanyl nucleotide-binding regulatory protein, Gs. Choleragen catalyzes the transfer of ADP-ribose from NAD to Gs which is thereby irreversibly activated. Bordetella pertussis toxin (PT) ADP-ribosylates Gi, abolishing the effects of inhibitory agonists and increasing basal adenylate cyclase activity. To study this apparently paradoxical stimulation of cyclase activity, NG108-15 (neuroblastoma x glioma hybrid) cells were incubated with PT or choleragen or with both toxins. Maximal cyclase activity observed following incubation with both toxins was greater than that seen following incubation with either toxin alone. This is consistent with the hypothesis that Gi exerts a basal inhibitory tone on the catalytic moiety which is relieved following ADP-ribosylation with PT. The amino acid acceptor for PT-catalyzed ADP-ribosylation is asparagine in transducin, a retinal protein structurally related to Gs and Gi. ADP-ribose-protein bonds have been described as stable or labile to neutral hydroxylamine. ADP-ribose-glutamate was thought to be the labile bond and ADP-ribose-arginine the stable bond. Study of the ADP-ribose-asparagine bond formed by pertussis toxin indicates that it represents a third class of ADP-ribose linkage which is even more stable to hydroxylamine degradation. The ADP-ribose-Gi linkage formed by PT is as stable as the ADP-ribose-transducin bond, suggesting that the PT substrate in Gi is asparagine. ADP-ribose bonds of similar stability are formed in membranes from human erythrocytes and NG108-15 cells after incubation with 32-P-NAD, suggesting the existence of tissue ADP-ribosyl-(asparagine)protein transferases in addition to the previously described ADP-ribosyl-(arginine)protein transferases.